An effective and safe gene delivery system is required for gene therapy to be clinically useful. Viral vectors are relatively efficient gene delivery systems, but suffer from a variety of limitations, such as the potential for reversion to the wild type as well as immune response concerns. As a result, nonviral gene delivery systems are receiving increasing attention (Worgall, et al., Human Gene Therapy 8:37-44 (1997); Peeters, et al., Human Gene Therapy 7:1693-1699 (1996); Yei, et al., Gene Therapy 1:192-200 (1994); Hope, et al., Molecular Membrane Biology 15:1-14 (1998)). Plasmid DNA-cationic liposome complexes are currently the most commonly employed nonviral gene delivery vehicles (Felgner, Scientific American 276:102-106 (1997); Chonn, et al., Current Opinion in Biotechnology 6:698-708 (1995)). However, complexes are large, poorly defined systems that are not suited for systemic applications and can elicit considerable toxic side effects (Harrison, et al., Biotechniques 19:816-823 (1995); Huang, et al., Nature Biotechnology 15:620-621 (1997); Templeton, et al., Nature Biotechnology 15:647-652 (1997); Hofland, et al., Pharmaceutical Research 14:742-749 (1997)).
Recent work has shown that plasmid DNA can be encapsulated in small (˜70 nm diameter) “stabilized plasmid-lipid particles” (SPLP) that consist of a single plasmid encapsulated within a bilayer lipid vesicle (Wheeler, et al., Gene Therapy 6:271-281 (1999)). These SPLPs typically contain the “fusogenic” lipid dioleoylphosphatidyl-ethanolamine (DOPE), low levels of cationic lipid, and are stabilized in aqueous media by the presence of a poly(ethylene glycol) (PEG) coating. SPLP have systemic application as they exhibit extended circulation lifetimes following intravenous (i.v.) injection, accumulate preferentially at distal tumor sites due to the enhanced vascular permeability in such regions, and can mediate transgene expression at these tumor sites. The levels of transgene expression observed at the tumor site following i.v. injection of SPLP containing the luciferase marker gene are superior to the levels that can be achieved employing plasmid DNA-cationic liposome complexes (lipoplexes) or naked DNA. Still, improved levels of expression may be required for optimal therapeutic benefit in some applications (see, e.g., Monck, et al., J. Drug Targ. 7:439-452 (2000)).
Typically, both liposomes and SPLPs comprise PEG-lipid derivatives. Typically, PEG-lipids are prepared by derivatization of the polar head group of a diacylglycerophospholipid, such as distearoylphosphatidylethanolamine (DSPE), with PEG. These phospholipids usually contain two fatty acyl chains bonded to the 1- and 2-position of glycerol by ester linkages. Unfortunately, these acyl groups are susceptible to cleavage under acidic or basic conditions. The resulting hydrolytic products, such as analogs of lysophospholipid and glycerophosphate, do not remain associated with the bilayer structure of the liposome or the SPLP. Unfortunately, such dissociation can weaken the integrity of the liposome or SPLP structure, leading to significant leakage of the bioactive agent or drug from the liposome or SPLP and contributing to instability during storage, and thus shortened shelf-life of the liposome or SPLP product. In addition, the loss of these hydrolysis products, such as PEG-lysophospholipid, from the liposome or SPLP negates the benefits otherwise resulting from the presence of the PEG-phospholipid.
Lipid stability is important in the development of liposomal or SPLP drug delivery systems. Therefore, it is desirable to develop PEG-lipids that are less susceptible to hydrolysis, thereby, increasing the circulation longevity of the liposomes or the SPLP. The present invention addresses this and other needs.